A large number of people suffer, or are predisposed to suffer from disturbances in their metabolism. One such disturbance includes insulin resistance, which is characteristic of the metabolic syndrome (syndrome X), polycystic ovary syndrome, obesity and type II diabetes, diseases that are rapidly growing in number in the western world. These diseases are multi-factorial and their mechanism or physiology are, in the majority of cases, not well characterized or understood. Type II diabetes includes the most prevalent form of diabetes, which results from insulin resistance with an insulin secretory defect. Pharmacological treatments such as metformin and rosiglitazone have an ameliorating effect on insulin resistance and are believed to increase the effectiveness of endogenous insulin and thereby contribute to the lowering of elevated blood glucose levels in type II diabetes patients.
One mechanism whereby insulin resistance may be induced is via elevation of reactive oxygen species (ROS). Although contrasting effects of ROS have been reported on the insulin signal transduction system and glucose transport, it has been shown that prolonged exposure of cells to ROS causes insulin resistance. Insulinomimetic effects of ROS have been reported using muscle cells and adipocytes. Acute exposure of adipocytes to H2O2 was shown to activate pyruvate dehydrogenase activity and lipid synthesis [May et al., Journal of Biological Chemistry, 254:9017–21 (1979)]. Some but not all aspects of insulin signaling appear to be activated by H2O2. Using L6 myocytes it was shown that H2O2 caused a PI3K-dependent activation of PKB and inhibition of GSK3 within 30 min of treatment [Tirosh et al., Journal of Biological Chemistry, 274:10595–602 (1999)]. Prolonged treatment (24 h) of L6 muscle cells and 3T3-L1 adipocytes with a ROS generating system increased the expression of GLUT1 that resulted in elevated basal glucose transport [Kozlovsky et al., Free Radical Biology & Medicine, 23:859–69 (1997); Kozlovsky et al., Journal of Biological Chemistry, 272:33367–72 (1997)]. Treatment of these cell lines with H2O2 also interferes with insulin signaling [Rudich et al., American Journal of Physiology, 272:E935–40 (1997)]. Simultaneous treatment with insulin and H2O2 was shown to inhibit insulin stimulated glucose transport and glycogen synthesis in spite of intact PKB activation [Blair et al., Journal of Biological Chemistry, 274:36293–9 (1999)]. Pretreatment with ROS inhibited insulin stimulated IRS-1 and PI3K cellular redistribution, PKB serine phosphorylation and glucose transport [Tirosh, Potashnik et al., Journal of Biological Chemistry, 274:10595–602 (1999)]. The antioxidant lipoic acid could prevent these effects [Rudich et al., Diabetologia, 42:949–57 (1999)]. Taken together, these results suggest that insulin signaling involve redox reactions, with some steps that can be mimicked and some that can be inhibited by H2O2. Integrating these findings with the demonstration that insulin can stimulate the production of H2O2, it can be hypothesized that ROS are involved in insulin signaling and may be responsible for the insulin resistance observed after prolonged treatment with insulin and other agents.
Oxidative stress is caused by excess free radical production in cellular metabolism. The free radicals derived from reaction products of oxygen are often termed reactive oxygen species (ROS). A reducing environment inside the cell prevents oxidative damage and can be maintained by the action of antioxidant enzymes and substances, such as superoxide dismutase (SOD), catalase, glutathione, selenium-dependent glutathione, thioredoxin hydroperoxidases, thioredoxin, vitamins C and E, and probably more unknown players.
Oxidative stress has been demonstrated in several different diseases and is implicated as an important driving force in the aging process [Finkel et al., Nature, 408:239–47 (2000); Spector, Journal of Ocular Pharmacology & Therapeutics, 16:193–201 (2000)]. A growing body of data demonstrate signs of increased oxidative stress in type II diabetes. It is likely that the oxidative stress is contributing to many of the vascular complications occurring in the late stages of the disease but the evidence for oxidative stress as causative factor in the development of insulin resistance and deterioration of beta cell function is still lacking. An inverse relationship between insulin action on glucose disposal and plasma superoxide ion, and a positive relationship between insulin action on glucose disposal and plasma GSH/GSSG ratio have been observed in type 2 diabetic patients during euglycemic hyperinsulinemic clamp [Paolisso et al., Metabolism: Clinical & Experimental, 43:1426–9 (1994)]. Decreased serum vitamin E content, a marker of impaired oxidant/antioxidant status, was reported to be associated with increased risk of developing type II diabetes [Salonen et al., BMJ, 311:1124–7 (1995)]. In animal experiments it was recently demonstrated that chemically induced oxidative stress exacerbated insulin resistance and hyperglycemia in obese Zucker rats [Laight et al., British Journal of Pharmacology, 128:269–71 (1999)]. There are also indications that beta cell toxic agents like alloxan and streptozotocin that are used to induce experimental animal diabetes act via oxidative stress [Davis et al., Biochemical Pharmacology, 55:1301–7 (1998); Hotta et al., Journal of Experimental Medicine, 188:1445–51 (1998)].
Superoxide can be produced by a number of cellular enzyme systems: NAD(P)H oxidases, xanthine oxidase, lipoxygenases, cyclooxygenase, P-450 monooxygenases, and the enzymes of mitochondrial oxidative phosphorylation. The majority of free radicals are produced by the mitochondria as unwanted by-products of the respiratory chain but the cell also purposely generates free radicals. The cellular defense system of the body utilizes oxygen radicals to kill invading microorganisms and the vascular system uses the nitric oxide radicals as an intermediate in the regulation of vascular tone. Originally, the NAD(P)H oxidase system responsible for production of superoxide that participates in bacterial killing was demonstrated in neutrophils and other phagocyte cells [Segal et al., Annals of the New York Academy of Sciences, 832:215–22 (1997)]. A growing number of experimental data from endothelial cells and other cell types show that ROS can be produced through activation of NAD(P)H-oxidase [Jones et al., American Journal of Physiology, 271:H1626–34 (1996); Krieger-Brauer et al., Journal of Biological Chemistry, 272:10135–43 (1997); Bayraktutan et al., Cardiovascular Research, 38:256–62 (1998)]. When activated, the NAD(P)H oxidase assembles at the plasma membrane and catalyses the single electron reduction of molecular O2 to superoxide (O2−) In the presence of superoxide dismutase, O2− dismutates to hydrogen peroxide (H2O2) that can be converted to a hydroxyl radical (OH−) in the presence of ferrous ions. The list of other free radicals originating from O2− that can be formed in the cell is longer, and will not be further discussed here. At least five proteins are required for the formation of an active NAD(P)H oxidase complex: the membrane bound cytochrome b558 and the cytosolic proteins, p47phox, p67phox, p40phox and a small GTP-binding protein, Rac-1 or Rac-2 [Abo et al., Journal of Biological Chemistry, 267:16767–70 (1992); Babior, Advances in Enzymology & Related Areas of Molecular Biology, 65:49–95 (1992); Knaus et al., Journal of Biological Chemistry, 267:23575–82 (1992)]. Cytochrome b558 is a flavoprotein with an NAD(P)H-binding site and consists of two subunits, gp91phox and p22phox [Sumimoto et al., Biochemical & Biophysical Research Communications, 186:1368–75 (1992)].
The hypoglycemic agent diphenylene iodinium (DPI) has been shown to diminish the rate of mitochondrial respiration by inhibiting NADH dehydrogenase. Holland et al. (1973; J. Biol. Chem. 248: 6050–6056) discloses that the enzyme inhibition causes the hypoglycemic action by decreasing mitochondrial oxidation and the hepatic and whole body ATP content (See also Gatley, S. J. & Martin, J. L. (1979) Xenobiotica 9: 539–546). However, it has not been previously shown that agents which inhibit NAD(P)H oxidase would be useful for increasing the activity of the insulin receptor and/or the intracellular insulin-signaling pathway, and thereby be useful against insulin resistance.